Method for culturing microalgae of the aurantiochytrium genus in a culture medium without chloride and without sodium for the production of DHA

Abstract

A method for culturing a protist of the Aurantiochytrium mangrovei genus. The genus is characterized genetically and by virtue of the lipid profile thereof. The method makes it possible to obtain a high biomass yield and a lipid, and more particularly docosahexaenoic acid (DHA), enrichment of the protists thus cultured. The development of a culture medium allows the production, at high cell density, of a DHA-rich protest of the Aurantiochytrium mangrovei genus. The medium is chemically defined at low sodium ion (Na + ) and chloride ion (Cl − ) content.

The invention claimed is: 
     
       1. A method for producing DHA in a culture of protists of the genus  Aurantiochytrium  comprising the following step:
 a) culturing, in heterotrophic or mixotrophic conditions, one or more strains of the genus  Aurantiochytrium  in a chemically defined culture medium, 
 b) maintaining said culture for several generations, and 
 c) recovering the biomass thus cultured, 
 wherein
 the one or more strains of the genus  Aurantiochytrium  has a genetic identity of at least 92% to sequence SEQ ID NO: 1 and 
 the chemically defined culture medium has less than 3.5 g/L of sodium ions and less than 1 g/L of chloride ions and has 200 mM to 500 mM organic carbon-containing substrate. 
 
 
     
     
       2. The method according to  claim 1 , wherein the strain of the genus  Aurantiochytrium  also has a genetic identity of at least 96% to sequence SEQ ID NO: 2, and/or at least 91% to sequence SEQ NO. 3 and/or at least 95% to sequence SEQ ID NO: 4. 
     
     
       3. The method according to  claim 1 , wherein the culture medium has less than 3.5 g/L of sodium ions and less than 200 mg/L of chloride ions. 
     
     
       4. The method of  claim 3 , wherein the culture medium has less than 1 g/L of sodium ions. 
     
     
       5. The method of  claim 3 , wherein the culture medium has less than 6 mg/L of sodium ions. 
     
     
       6. The method according to  claim 1 , wherein the medium does not contain an osmotic pressure regulatory agent. 
     
     
       7. The method according to  claim 1 , further comprising the steps of:
 d) recovering the lipids of the strains, and optionally, 
 e) extracting the DHA (docosahexaenoic acid). 
 
     
     
       8. The method according to  claim 1 , wherein the culture medium consists of: 
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   Ingredients 
                   Concentration 
                 
                     
                     
                 
                     
                 
                 
                 
                 
                 
               
                     
                   KCl 
                   0.05-5  
                   g/L 
                 
                     
                   H 3 BO 3   
                   0.01-0.3  
                   g/L 
                 
                     
                   MgSO 4 , 7H 2 O 
                   2-10  
                   g/L 
                 
                     
                   CaCl 2 , 2H 2 O 
                   0.2-0.9  
                   g/L 
                 
                     
                   KNO 3   
                   0.01-0.06  
                   g/L 
                 
                     
                   KH 2 PO 4 , 7H 2 O 
                   0.2-1  
                   g/L 
                 
                     
                   Na 2 EDTA, 2H 2 O  
                   0.001-0.005  
                   g/L 
                 
                     
                   ZnSO 4 •7H 2 O 
                   0.01-0.1  
                   mg/L 
                 
                     
                   CoCl 2 •6H 2 O 
                   0.01-0.1  
                   mg/L 
                 
                     
                   MnCl 2 •4H 2 O 
                   0.05-1  
                   mg/L 
                 
                     
                   Na 2 MoO 4 , 2H 2 O  
                   0.0005-0.1  
                   mg/L 
                 
                     
                   Na 2 SeO 3   
                   0.01-0.5  
                   mg/L 
                 
                     
                   NiSO 4 •6H 2 O 
                   0.5-5  
                   mg/L 
                 
                     
                   CuSO 4 •5H 2 O 
                   0.0025-1  
                   mg/L 
                 
                     
                   EDTA—Fe 
                   10-50  
                   mg/L 
                 
                     
                   Glucose 
                   20-60  
                   g/L 
                 
                     
                   (NH 4 ) 2 SO 4   
                   2-9  
                   g/L 
                 
                     
                   Thiamine 
                   1-50  
                   mg/L 
                 
                     
                   Vitamin B12 
                   0.025-5  
                   mg/L 
                 
                     
                   Pantothenate 
                   0.1-25  
                   mg/L. 
                 
                     
                     
                 
             
                
                
                
               
               
                
               
            
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
       9. The method according to  claim 1 , wherein the biomass derived from step b) represents at least 100 g/L of dry matter. 
     
     
       10. The method according to  claim 1 , wherein the DHA concentration at the conclusion of step b) represents at least 15 g/L. 
     
     
       11. The method according to  claim 1 , wherein the DHA contained in the biomass at the conclusion of step b) represents more than 30% of the total lipids. 
     
     
       12. The method according to  claim 1 , wherein it has a DHA productivity of at least 0.1 g/L/h. 
     
     
       13. The method according  claim 1 , wherein said organism of the genus  Aurantiochytrium  corresponds to strain FCC 1324, deposited with the CCAP (Culture Collection of Algae and Protozoa), under accession number CCAP 4062/1. 
     
     
       14. The method according to  claim 1 , wherein the medium does not contain an osmotic pressure regulatory agent selected among the group consisting of mannitol, sorbitol, polyethylene glycol and sucrose.